线粒体CypD-Poldip 2轴参与脑缺血再灌注损伤新机制及其阻断剂发现

The crosstalk between mitochondria and NADPH oxidases (NOX) exist in a feed-forward regulation of the ROS sources during cerebral ischemia-reperfusion insult. Knowledge of the mechanism will further contribute in offering novel therapeutic targets against ischemic stroke. The findings of our group first showed that polymerase δ-interacting protein 2 (Poldip 2), a newly discovered NOX 4 enhancer protein, accumulates in the mitochondrial matrix and interaction with the cyclophilin D (CypD) during oxidative stress. In contrast, cyclosporine A (CsA), a CypD inhibitor, pretreatment prevents this complex, inhibits excessive NOX 4 activation, and decreases ROS generation, indicating that CypD-Poldip 2 axis probably as a novel pathway involved in cerebral ischemia reperfusion. However, as an immunosuppressant drug, the serious adverse reaction of CsA limits its clinical application in ischemic stroke. Molecular docking and previous experimental data suggest that the neuroprotective effect of GA probably related to inhibit CypD-Poldip 2 axis, which deserves further assessment. To test this idea, The novel mechanisms of cerebral ischemia reperfusion injury based on mitochondrial CypD-Poldip 2 axis and inhibitor GA will be studied in this project at the molecular, subcellular, cellular, and animal levels.

脑缺血再灌注条件下线粒体与NADPH氧化酶（NOX）在活性氧自由基（ROS）生成前馈调节中存在crosstalk，认识该机制有助于找到治疗缺血性脑中风的新靶点。本课题组首次发现：氧化应激损伤后NOX 4调节蛋白——σ整合蛋白聚合酶2 （Poldip 2）与亲环素D（CypD）结合并在线粒体中聚集，而CypD抑制剂环孢素A（CsA）可阻断该过程，抑制线粒体NOX 4激活与ROS生成，提示CypD-Poldip 2轴可能是调节脑缺血再灌注损伤的新途径。作为一种免疫抑制剂，CsA在治疗脑中风临床应用中受到限制。计算机分子对接与前期试验结果提示没食子酸（GA）的神经保护作用可能与抑制CypD-Poldip 2轴有关，其机制值得进一步研究。为验证以上假说，本课题将从分子、亚细胞、细胞和动物四个水平，阐明CypD-Poldip 2轴参与脑缺血再灌注损伤及GA阻断CypD-Poldip 2轴的分子机制。

脑缺血再灌注损伤条件下线粒体与NADPH氧化酶（NOX）在活性氧自由基（ROS）生成前馈调节中存在重大关联，认识该机制有助于找到治疗缺血性脑中风的新靶点。本课题组分别从动物、细胞、细胞器、分子四个层面，对亲环素D（CypD）-σ整合蛋白聚合酶2（Poldip 2）轴调控脑缺血再灌注中线粒体Poldip 2 聚集，CypD-Poldip 2 轴调控线粒体NOX 4 活力与线粒体功能紊乱，以及没食子酸（GA）阻断线粒体氧化应激损伤机制，三大问题进行了系统研究。本课题组首次发现Poldip 2蛋白并未在脑缺血再灌注损伤时的线粒体中聚集，而是伴随线粒体通透孔（MPTP）开放所致线粒体内钙超载，发生了钙依赖性构象改变，而Poldip 2蛋白构象改变是其在CypD介导下与NOX 4结合并使其活化产生线粒体氧化应激损伤的关键因素之一。此外本课题组还明确了GA 的分子靶点为CypD，其干预NOX 4活化的分子机制为调控CypD的功能，关闭MPTP，抑制线粒体钙超载，从而阻断Poldip 2构象改变以及NOX 4的活化，有效干预了线粒体氧化应激损伤。GA将有望成为治疗缺血性脑中风的新型药物，其具体成药性和临床治疗效果也值得进一步研究。